Immunohistochemistry Guide

Making IHC as easy as ABC

Table of Contents
Helping you to reach new visualization frontiers in your research: 2 Introduction
Vector Laboratories was founded on a growing
this is our mission. Since our founding in 1976, a primary driving 2 Immunohistochemistry Workflow portfolio of purified lectins and lectin conjugates
principle has been to develop and manufacture labeling and 4 Immunohistochemistry Selection Guide that helped to pioneer lectin histochemistry.
These products remain a key component of our
detection technologies that make IHC as easy as ABC. 6 Pioneering in IHC Technology business today. In the early 1980s, we leveraged

A.
our expertise in histochemistry to revolutionize
Reliable and reproducible reagents that instill trust 8 Choosing a Detection System the field of IHC with the commercialization of
antibody-based avidin-biotin reagents and the
and confidence. 10 Avidin-Biotin (ABC) Based Detection introduction of the VECTASTAIN® ABC system.
VECTASTAIN® ABC Systems

B.
This system enabled routine laboratory use of
Simple and robust product designs that streamline workflows – VECTASTAIN® ABC Kits IHC with any standard brightfield microscope.
Following the success of the ABC kits, Vector
and allow elucidation of complex biological systems. – Choosing a VECTASTAIN® ABC Kit Laboratories has continued to introduce many

C.
– Customizing your VECTASTAIN® ABC Kit novel and innovative products to support
A knowledge base of over 100 years of combined IHC research endeavors for cell and tissue antigen
18 Polymer-Based Detection visualization. These include the ImmPRESS™
experience to help you accelerate the pace of discovery. – ImmPRESS™ One-Step Polymer Systems micropolymer reagents, Mouse on Mouse
– ImmPRESS™ Two-Step Amplified Polymer Systems (M.O.M.™) detection systems, unique ImmPACT™
It’s as simple as that. enzyme substrates, and VECTASHIELD® antifade
– Choosing an ImmPRESS™ Polymer Kit mounting media for fluorescent applications.
– Multiple Antigen Labeling Simplified
Mouse Colon: Smooth Muscle Actin (m), M.O.M.™ Basic Kit, VECTASTAIN® ABC-AP Kit, Vector® Red (magenta).
24 Species on Species Detection (Mouse)
25 Mouse on Mouse (M.O.M.™) Immunodetection Kits

26 Choosing an Enzyme Substrate

– Enzyme Substrates
– Enzyme Substrate Properties

30 Multiple Antigen Labeling

– Enzyme Substrate Combinations

34 Counterstaining
– Counterstain/Substrate Compatibility

36 Blocking Background Signal

– BLOXALL™ Endogenous HRP/AP Blocking Solution
– Avidin/Biotin and Streptavidin/Biotin Blocking Reagents
– Normal Sera
– Animal-Free Blocking Solutions
– General Protein Blocking Reagents
38 Secondary and Tertiary Detection Reagents
– Biotinylated and Unconjugated Secondary Antibodies
– Enzyme Conjugated Secondary Antibodies
– Avidin and Streptavidin Enzyme Conjugates
40 Mounting Media
– VectaMount™ Mounting Media
Front cover: Mouse liver section in which human
colorectal cancer-derived organoids have spread.
This liver metastasis shows the invading tumor
front (keratin-20, brown tumor cell) growing
from the top of the images towards the bottom,
embedded in increasing surrounding connective
tissue (fibronectin, green fibers). All nuclei of
human tumor cells and the mouse hepatocytes
in the lower part of the image were stained with
a nuclear membrane marker (lamin A, red round
shapes). Slight green staining in the lower part of
the image shows connective tissue that surrounds
blood vessels in normal liver tissue. (Image
provided by Dr. Steffen Rickelt and Dr. Jatin Roper,
Massachusetts Institute of Technology, USA).

42 Accessory Reagents
– VECTABOND™ Reagent Tissue Section Adhesive
– ImmEdge™ Hydrophobic Barrier Pen
– Histology Pens, Control Antibodies, and Antigen Unmasking Solutions

vectorlabs.com vectorlabs.com
 Immunohistochemistry Workflow
Vector Laboratories is your resource for premium labeling and detection products
at each step of your IHC workflow.

Tissue Antigen Quench/ Primary Secondary Tertiary Substrate/ Coverslip/

Antibody/ Counterstain Visualize
Preparation Retrieval Block Antibody Reagent Chromogen Mount
Lectins*

• VECTABOND™ • Antigen Unmasking • BLOXALL™ • Biotinylated • ImmPRESS™ • VECTASTAIN® ABC • HRP substrates • Hematoxylin • VectaMount™
Tissue Section Solutions, Citrate- Endogenous HRP lectins Polymer Reagents complex reagents Mounting
Adhesive or Tris-based and AP Blocking (HRP or AP) • AP substrates/ • Methyl Green Medium
Solution • Unconjugated • ImmPRESS™ Levamisole
• ImmPrint™ lectins • Mouse on Mouse Excel Amplified Solution • Nuclear Fast Red • VectaMount™ AQ
Histology Pen • Avidin/Biotin (M.O.M.™) ImmPRESS™ Staining Kits Mounting
Blocking Kit Polymer Kit Medium
• ImmEdge™ • Mouse on Mouse
Hydrophobic • Streptavidin/Biotin • Mouse on Mouse (M.O.M.™) Elite® Kit
Barrier Pen Blocking Kit (M.O.M.™) Basic Kit (HRP)

• Normal Sera • Biotinylated • Enzyme-conjugated

secondary antibodies avidin/streptavidin
• Animal-Free Block (HRP or AP)
and Diluent • Unconjugated
secondary antibodies
• BSA
• Enzyme-conjugated
• Casein Solution secondary antibodies
(HRP or AP)
• Vector® M.O.M.™
Mouse Ig Blocking • Biotinylated
Reagent anti-lectins

• Carbo-Free Block

* For more information visit: vectorlabs.com/lectins

2 vectorlabs.com vectorlabs.com 3
 Immunohistochemistry Selection Guide
Follow the simple steps below to choose the most appropriate labeling and detection solution
for your experiment.

Legend
Choose Primary Antibody Blocking Reagents Choose Enzyme Substrate
1 • Specific for antigen of interest
• Choices determined by the options
selected in Steps 1-4
3 • Color
• Consider tissue species and • BLOXALL™ Endogenous HRP and AP
• Compatibility with other system reagents
preparation (fixation) Blocking Solution (counterstains, mounting media and other
Primary
• Consider antigen retrieval • Avidin/Biotin Blocking Kit substrates for multiplexing) antibody
(if using VECTASTAIN® ABC system)
requirements
• Normal Sera (from the species of
secondary antibody)
• M.O.M.™ Mouse Ig Blocking Reagent
• Animal-Free Block and Diluent Secondary
VECTABOND™ Reagent • BSA antibody
(Tissue Section Adhesive) • Casein Solution Choose Nuclear Counterstain
4 • Blue, green, or red
SECONDARY DETECTION SYSTEMS • Compatibility with substrate,
One Step mounting media Amplifier
Choose Secondary Antibody
2 and Tertiary Detection System
Option A
Convenient. Consistent. Ready-to-use.
Non-Biotin based.
antibody

• Choose HRP or AP enzyme system • ImmPRESS™ Polymer Reagents

• Consider sensitivity requirements
• Consider species of primary antibody Enzyme
(HRP or AP)
• Consider tissue species
or Choose Mounting Media
5 • Aqueous vs. non-aqueous
Two Step • Compatibility with substrate(s) Biotinylated
Option B
Economical. Biotin-based. and counterstain enzyme
(HRP or AP)
• Biotinylated secondary antibody
+ ABC Complex
(VECTASTAIN® Elite® ABC Kit)

Avidin

or
Visualize
Option C
Two Step 6 • Brightfield microscope
Highest sensitivity. Non-biotin based.
• ImmPRESS™ Excel Amplified
Polymer Staining Systems

Small bowel: CD10 (m), VECTASTAIN® Elite® ABC Kit,

ImmPACT™ DAB HRP Substrate (brown). Hematoxylin
QS counterstain (blue).

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 Pioneering in IHC Technology
Observation is one of the fundamental steps in the scientific
method. However, for centuries the scientific study of tissues
was limited to observations of dissections with the unaided
eye (gross anatomy).
This all changed in the 17th century when Anton Van
Leeuwenhoek fabricated a microscope that allowed
observations of tissues at the cellular level, thus establishing
the science of histology. While early researchers found it
relatively simple to distinguish between the cell boundaries
and subcellular compartments in plants, doing so in animal
tissue presented a much greater challenge. It wasn’t until
the late 19th century with the introduction of dyes, such as
hematoxylin that Paul Mayer used to successfully stain nuclei,
that the subcellular structure of tissues became visible and
the science of histochemistry emerged.
The number of available tissue dyes and stains increased
during the early 20th century, as did the number of molecular
families they identified. However, the ability to identify
individual cellular- or tissue-specific proteins remained elusive.
This changed in the mid-20th century when Dr. Albert Coons
demonstrated that fluorescently labeled antibodies could be
used to localize bacteria inside macrophages, thus helping to
pioneer the science of immunohistochemistry (IHC). Over the
next two decades our understanding of antibodies, antigens
and immunology grew rapidly. However, IHC remained
largely a specialized research tool used primarily in university
settings. Then in the late 1960’s, Dr. Stratis Avrameas and
Dr. Paul Nakane independently developed methods to
covalently couple the enzyme horseradish peroxidase (HRP)
to antibodies. HRP in the presence of diaminobenzidine and
hydrogen peroxide creates a brown precipitate at the site of
the HRP-labeled antibody. The precipitate can be visualized
using an ordinary light microscope. This allowed for the IHC
results to be viewed in any lab having a light microscope,
with no need for expensive, complicated fluorescence
instrumentation.
The use of IHC as a research tool grew dramatically over
the next decade. The technique began to be used in clinical
settings at large university hospitals. The HRP assay system
was further improved in the early 1980’s when Dr. Su-Ming
Hsu showed that the high affinity of avidin for biotin could be
used to increase the stability of the enzyme antibody complex
and improve the sensitivity of the assay. Vector Laboratories
was instrumental in the development of the IHC field by
commercializing such key technologies. The use of avidin- and
biotin-based detection systems dominated the IHC market for
the next two decades.
During this time Dr. Shan-Rong Shi introduced “antigen
retrieval” for formaldehyde-fixed tissues. This technique
allowed IHC to be readily performed on formalin-fixed,
paraffin-embedded tissues, greatly increasing clinical utility of
IHC. However, in addition to improving antigenicity in tissue
sections, antigen retrieval also exposed numerous sites of
endogenous biotin that were previously undetected. This
required steps to be added to IHC staining protocols to block
endogenous biotin in biotin-containing specimens. In clinical
settings in particular, antibody detection strategies returned
to non-biotin HRP systems to avoid confusion resulting from
endogenous biotin. However, the choice was not clear-cut, as
avidin-biotin detection systems offered greater sensitivity than
the previous peroxidase-labeled antibody systems.
This dilemma was finally resolved in the mid-2000s by the
emergence of biotin-free polymer/multimer detection
systems that offered similar sensitivity to avidin-biotin
detection systems. Although early polymer-based systems
suffered from background and tissue penetration problems,
today’s systems deliver performance comparable to the best
avidin-biotin detection systems.
References
Coons AH, Creech HJ and Jones RN “Immunological properties of an
antibody containing a fluorescent group” Proc. Soc. Exp. Biol. Med. 47,
200-202 (1941)
Nakane P and Pierce GB Jr “Enzyme-labeled antibodies for the light and
electron microscopic localization of tissue antigens” J. Cell. Biol. 33,
307-318 (1967)
Leduc E, Avrameas S and Bouteille M “Ultrastructural localization of
antibody in differentiating plasma cells” J. Exp. Med. 127, 109-118. (1968)
Hsu S-M, Raine L, and Fanger H “Use of Avidin-Biotin-Peroxidase Complex
(ABC) in Immunoperoxidase Techniques: A Comparison between ABC and
Unlabeled Antibody (PAP) Procedures” J. Histochem. Cytochem. 29(4),
577-580 (1981)
Shi SR, Key ME and Kalra KL “Antigen retrieval in formalin-fixed, paraffin-
embedded tissues: an enhancement method for immunohistochemical
staining based on microwave oven heating of tissue sections” J Histochem
Cytochem. Jun, 39(6), 741-8 (1991)
Childs GV “History of Immunohistochemistry” Pathobiology of Human
Disease. 3775-3796 (2014)

1941 1980 1991 1999 2005 2011 2016

Albert Coons demonstrated that
fluorescently labeled antibodies can
be used to localize bacteria inside
macrophages (immunohistochemistry).
Vector Labs first to commercialize S.R. Shi developed the Vector Labs introduced Vector Labs developed Vector Labs introduced Strategic investment by
avidin-biotin enzyme complex antigen retrieval process for a next-generation PAP detection reagents enabling BLOXALL® Endogenous HRP Maravai LifeSciences
(VECTASTAIN® ABC kits) formaldehyde fixed tissues pen conforming with greater access to antigens and AP Blocking Solution
environmental regulations within tissues and
(ImmEdge™ Pen) improving multiple antigen
labeling (ImmPRESS™
enzyme polymer)

1966 1981 1986 1993 2004 2007 2014

Stratis Avrameas and
Paul Nakane independently
developed methods to
covalently couple the enzyme
horseradish peroxidase (HRP)
to antibodies
S.M. Hsu published that the high
affinity of avidin for biotin could
be used to increase the stability
of the enzyme antibody complex
Vector Labs launched
PHOTOPROBE® Biotin
Vector Labs introduced first Vector Labs expanded antifade Vector Labs introduced Vector Labs introduced
antifade mounting media mounting media with hardening proprietary substrates additional proprietary AP
for fluorescence formulation (VECTASHIELD® enabling multiplexing substrate (ImmPACT™ Vector®
(VECTASHIELD® HardSet™ Mounting Media) IHC (ImmPACT™ HRP Red substrate)
Mounting substrates)
Media)

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 Choosing a Detection System Comparison of Detection Systems
Immunohistochemistry Overview Choose the appropriate detection system for your experiment based on enzyme, sensitivity, cost,
biotin vs. non-biotin formats, flexibility, and time considerations.
Immunohistochemistry (IHC) is a method to detect specific target antigens
(proteins) in tissue sections using antibodies. Immunocytochemistry (ICC)
Mouse
uses similar techniques to localize cellular proteins in cell preparations. Primary Ready-to- Typical
Both IHC and ICC are powerful tools that provide insights into gene Cost/ Biotin- Micro- on Mouse Use (R.T.U.) number
expression, spatial relationships, and biomarker identification in a Detection System Enzyme Sensitivity Assay Free polymer Modular Tissue Format of steps

wide variety of applications. These applications include basic research, ImmPRESS™ Kits

assessment of normal and disease states within human and animal tissues, ImmPRESS™ Excel Amplified HRP Polymer Kits HRP •••••• •••• • • • 2
and assessment of plant pathology. ImmPRESS™ HRP Polymer Kits HRP ••••• ••• • • • 1
ImmPRESS™ VR HRP Polymer Kits HRP ••••• ••• • • • 1
The target antigen, bound by the detection antibody, is visualized using
ImmPRESS™ AP Polymer Kits AP ••••• ••• • • • 1
either chromogenic or fluorescence detection. In chromogenic detection,
the detection antibody is conjugated to an enzyme. The enzyme, usually VECTASTAIN® Kits

horseradish peroxidase or alkaline phosphatase, catalyzes the conversion VECTASTAIN® Elite ® ABC Kits HRP ••••• •• • 2

of its respective chromogen to a colored precipitate at the site of the R.T.U. VECTASTAIN® Elite ® Kits HRP ••••• •• • • 2
antigen. This precipitate can be visualized by using brightfield microscopy. VECTASTAIN® Universal Quick Kits HRP •••• •• • 2
Certain chromogens can also be visualized by using electron, darkfield or R.T.U. VECTASTAIN® Universal Quick Kits HRP •••• •• • • 2
fluorescence microscopy. In fluorescence detection, the detection antibody
VECTASTAIN® ABC-AP Kits AP •••• • • 2
is conjugated to a fluorophore which can be visualized using fluorescence
Original VECTASTAIN® ABC Kits HRP ••• • • 2
microscopy.
Mouse on Mouse (M.O.M.™) Kits
For the purposes of this guide IHC will be referenced for both IHC and Mouse on Mouse (M.O.M.™) ImmPRESS™ HRP ••• ••• • • • 1
ICC techniques. Polymer Kit
Mouse on Mouse (M.O.M.™) Kits HRP ••• ••• • • 2
Additional Options
Lymph Node: • Ki67 (m), VECTASTAIN® Universal ABC-AP Kit, Vector® Red Substrate (red)
• Multi-cytokeratin (m), VECTASTAIN® Universal Elite® ABC Kit, DAB substrate (brown). Enzyme Conjugated Avidin/Streptavidin HRP ••• • • 2
or AP
R.T.U. HRP Avidin/Streptavidin HRP ••• • • • 2
Enzyme Conjugated Secondary Antibody HRP •• • • 1
or AP

HRP - Horseradish peroxidase

AP - Alkaline phosphatase
VR - Veterinary Reagents

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 Choosing a Detection System

Avidin-Biotin Complex (ABC)-Based Detection VECTASTAIN® ABC Systems

Modular and versatile with high sensitivity and low background VECTASTAIN® ABC detection systems are uniquely formulated with our Avidin DH
Recommended applications:
and biotinylated enzyme conjugates to deliver enhanced signal sensitivity with low
ABC-based detection is one of the most widely-used methods for staining. These systems exploit the high affinity exhibited background. They are compatible with a wide range of target types, applications, and
Tissue and cell staining
between the protein avidin and the vitamin biotin. Avidin is tetravalent, so each avidin molecule can bind up to four biotinylated substrates. These reliable and economical VECTASTAIN® ABC Systems have come to be
conjugates. In ABC systems, avidin and biotinylated enzyme is combined to form large macromolecular complexes containing a mainstay product in immunohistochemistry laboratories. Protein and nucleic
multiple enzyme molecules. These added complexes bind to any biotinylated target, such as primary or secondary antibodies, acid blotting
nucleic acids, lectins, and macromolecules. When the chromogenic enzyme substrate is applied, it yields a colored precipitate at In situ hybridization
the site of the reaction. The large multi-enzyme complexes amplify the signal, providing greater sensitivity.
ELISAs
Neuronal tracing

Using the VECTASTAIN® ABC System

1 Add Primary Antibody 2 Add Biotinylated Secondary Antibody

3 Add Avidin/Biotinylated Enzyme Complex (ABC) 4 Add Enzyme Substrate

Tumor: • p53 (m), VECTASTAIN® Elite® ABC Kit, Vector® NovaRED™ (red) • Cytokeratin (s), VECTASTAIN® Elite® ABC Kit, Vector® SG (blue-gray).

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 Avidin-Biotin Complex (ABC)-Based Detection

VECTASTAIN® ABC Kits

Peroxidase-Based Kits: VECTASTAIN® Elite® ABC Kits
VECTASTAIN® Universal Quick Kits (Peroxidase) VECTASTAIN® Quick Kits
Peroxidase substrates produce sharp, dense precipitates with crisp localization. (Peroxidase) With VECTASTAIN® Universal Quick Kits, you can quickly detect primary antibodies (Peroxidase)
In conjunction with the high sensitivity and low background of VECTASTAIN® ABC made in mouse, rabbit, or goat. These kits rely on a proprietary preformed peroxidase-
Systems, peroxidase-based detection systems are a preferred choice for many Standard (Elite® ABC Reagent streptavidin complex to achieve outstanding sensitivity with short incubation times. R.T.U. VECTASTAIN Quick Kit,
applications. (For peroxidase substrates see p. 26-29.) only) Universal
• Rapid protocol: Staining in less than 20 minutes following primary antibody
Rabbit IgG incubation. Working solutions can be used immediately after dilution. VECTASTAIN Quick Kit,
Universal
VECTASTAIN® Elite ® ABC System (Peroxidase) Mouse IgG • High sensitivity, low background
The VECTASTAIN® Elite ® ABC complex is smaller and more uniform than conventional Human IgG • Biotinylated Universal Pan-Specific secondary antibody recognizes mouse, rabbit, and
avidin-biotin complexes. They have greater accessibility to biotinylated targets within Rat IgG goat primary antibodies, as well as those from related species such as rat, bovine,
tissue samples. VECTASTAIN® Elite ® ABC Kits are our most sensitive avidin-biotin based and sheep. (Do not use to stain rat, mouse or other rodent, rabbit, goat, bovine, or
Goat IgG
peroxidase systems. sheep tissue due to potential reactivity with endogenous IgG.)
Sheep IgG
• Highest available sensitivity, low background • Available in concentrate or ready-to-use format
Universal
• Cost effective: Higher sensitivity means lower cost per slide
R.T.U. VECTASTAIN® Elite® ABC
• Available without (Standard Kit) or with biotinylated species-specific or universal
Reagent Alkaline Phosphatase-Based Kits:
secondary antibodies
R.T.U. VECTASTAIN® Elite® ABC Kit, The sensitivity of the VECTASTAIN® ABC-AP system is comparable to that of the
• Available in ready-to-use formats that yield the same high sensitivity and low peroxidase VECTASTAIN® Elite® ABC system. You may prefer to use VECTASTAIN® ABC-AP VECTASTAIN® ABC-AP Kits
background as the corresponding conventional VECTASTAIN® ABC Kit reagents. (Alkaline Phosphatase)
Kits for tissues that have high endogenous peroxidase activity. The system also offers
additional substrate color choices. (For alkaline phosphatase substrates see p. 26-29.) Standard (ABC Reagent only)
VECTASTAIN® ABC Kits
Original VECTASTAIN® ABC Kit (Peroxidase) (Peroxidase) Rabbit IgG
Our original avidin-biotin ABC complex formulation. Mouse IgG
Standard (ABC Reagent only)
• Good sensitivity, low background Mouse IgM
Rabbit IgG
• Available with or without biotinylated species-specific secondary antibody Human IgG
Mouse IgG
• Economical Rat IgG
Mouse IgM
Human IgG Goat IgG

Rat IgG Sheep IgG

Goat IgG Universal

Sheep IgG
Guinea Pig IgG

Product Elite® (Peroxidase) Original (Peroxidase) Quick (Peroxidase) Alkaline Phosphatase

Note: Species-specific kits are selected corresponding to VECTASTAIN® ABC Kit, Standard PK-6100 PK-4000 AK-5000
the species in which the primary antibody is raised.
VECTASTAIN® ABC Kit, Rabbit IgG PK-6101 PK-4001 AK-5001
VECTASTAIN® ABC Kit, Mouse IgG PK-6102 PK-4002 AK-5002
VECTASTAIN® ABC Kit, Mouse IgM PK-4010 AK-5010
VECTASTAIN® ABC Kit, Human IgG PK-6103 PK-4003 AK-5003
VECTASTAIN® ABC Kit, Rat IgG PK-6104 PK-4004 AK-5004
VECTASTAIN® ABC Kit, Goat IgG PK-6105 PK-4005 AK-5005
VECTASTAIN® ABC Kit, Sheep IgG PK-6106 PK-4006 AK-5006
VECTASTAIN® ABC Kit, Guinea Pig IgG PK-4007
VECTASTAIN® ABC Kit, Universal PK-6200 AK-5200
R.T.U. VECTASTAIN® ABC Reagent PK-7100
R.T.U. VECTASTAIN® ABC Kit, Universal PK-7200

Tonsil: • Multi-Cytokeratin (m), VECTASTAIN® Elite® ABC Kit, Vector® DAB (brown) • CD3 (m), VECTASTAIN® Elite® R.T.U. VECTASTAIN® Universal Quick Kit PK-7800
ABC Kit, Vector® VIP (purple) • CD20 (m), VECTASTAIN® Elite® ABC Kit, Vector® SG (blue-gray). VECTASTAIN® Universal Quick Kit (concentrate) PK-8800

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 Avidin-Biotin Complex (ABC)-Based Detection

Choosing a VECTASTAIN® ABC Kit

Choose the Choose the Choose a

detection enzyme appropriate ABC kit convenient format

• Peroxidase To detect a biotinylated target, VECTASTAIN® ABC Kits are

• Alkaline phosphatase you will need: available in economical
• VECTASTAIN® ABC Reagent contained concentrated formats.
in the standard VECTASTAIN® ABC Kit For additional convenience and
ease of use some peroxidase-based
• An appropriate substrate kits are offered in ready-to-use
prediluted stabilized formats.
To detect an unlabeled primary antibody
or lectin, you will need:
• A biotinylated secondary antibody that
binds to the primary antibody species
or lectin you have chosen
• VECTASTAIN® ABC Reagent
• An appropriate substrate

For example, to detect a primary

antibody made in rabbit, the appropriate
choice is a VECTASTAIN® ABC Kit
designated Rabbit IgG.
Tumor: • Ki67 (m), VECTASTAIN® Elite ® ABC Kit, Vector® DAB (brown) • CD34 (m), VECTASTAIN® ABC-AP Kit, Vector® Blue (blue) • Cytokeratin AE1/AE3
(m), VECTASTAIN® ABC-AP Kit, Vector® Red (red).

Consider Species Cross-Reactivity

When choosing the optimal detection system for your • Use a biotinylated secondary antibody specifically adsorbed
application, it is important to consider not only the species of to remove cross-reacting antibodies of closely-related
the primary antibody but also the species of the tissue under species (e.g. biotinylated anti-mouse IgG, rat adsorbed).
examination. If the species of the primary antibody and the • Use the Mouse on Mouse (M.O.M.™) Immunodetection
species of the tissue are closely related (for example, rat and System for applications of mouse primary antibodies on
mouse), the biotinylated secondary antibody may cross-react mouse tissue (p. 24-25).
with endogenous IgG in the tissue section. This can lead to
background staining.
Substrates
The following options may minimize background staining in After choosing the VECTASTAIN® ABC Kit for your application,
these instances: select a substrate that matches the enzyme system of the kit
• Directly label the primary antibody with biotin (ProtOn™ (p. 26-29).
Biotin Labeling Kit, PLK-1202) and detect it using the
VECTASTAIN® Elite® ABC Kit (Standard – PK-6100, includes
VECTASTAIN® Elite® ABC Reagent only) followed by an
HRP substrate. Alternatively, label the primary with HRP
(ProtOn™ HRP Labeling Kit, PLK-1203) and develop with
an HRP substrate.

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 Avidin-Biotin Complex (ABC)-Based Detection

Customizing your VECTASTAIN® ABC Kit

Endometrium: • Progesterone Receptor (rm), VECTASTAIN® Universal ABC- AP Kit,
Vector® Blue AP Substrate (blue) • CD34 (m), VECTASTAIN® Universal Elite ® ABC Kit,
Vector® DAB HRP Substrate (brown).
If a VECTASTAIN® ABC system is not available with a biotinylated secondary antibody of your required specificity, you
can custom-build the exact kit that you require. All of our biotinylated, affinity-purified secondary antibodies (p. 38-39)
are designed for use with VECTASTAIN® ABC Standard Kits and the appropriate blocking serum. Our mix-and-match kit
components allow you to both design a custom kit to suit your needs and to use kit components interchangeably.
The reagents can be purchased individually, allowing you to combine them to suit your specific needs.
For example, to make a VECTASTAIN® Elite ® ABC Kit for use with a mouse IgG primary antibody on rat tissues:
1. Choose the VECTASTAIN® ABC Kit that contains the desired detection enzyme but with no secondary antibody
(e.g. VECTASTAIN® Elite ® ABC Kit, Standard).
2. Choose the biotinylated secondary antibody (e.g. biotinylated horse anti-mouse IgG, rat adsorbed).
3. Choose the blocking solution. We recommend a serum from the same species as the secondary antibody. (In our
example, normal horse serum). Alternatively, select our animal-free blocking reagents for multiple antigen labeling
(multiplex) IHC applications where antibodies from different species and a variety of detection systems are used
Tumor: • p53 protein (m), VECTASTAIN® ABC-AP Kit, Vector® Red AP Substrate (red)
• Pan-Cytokeratin (sheep), VECTASTAIN® Elite ® ABC Kit, Vector® SG HRP Substrate
on the same tissue section.
(blue/gray).
1. Choose Standard VECTASTAIN® ABC Kit with the appropriate detection enzyme

Enzyme Product Catalog Number

Peroxidase VECTASTAIN® Elite ® ABC Kit PK-6100
Peroxidase R.T.U. VECTASTAIN® Elite ® ABC Reagent PK-7100
Peroxidase VECTASTAIN® ABC Kit PK-4000
Alkaline Phosphatase VECTASTAIN® ABC-AP Kit AK-5000

2. Choose the biotinylated secondary antibody*

Product Concentrate R.T.U.†
Anti-Goat IgG (H+L) made in rabbit, biotinylated BA-5000
Anti-Goat IgG (H+L) made in horse, biotinylated BA-9500 BP-9500
Anti-Human IgG (H+L) made in goat, biotinylated BA-3000
Anti-Mouse IgG (H+L) made in horse, biotinylated BA-2000 BP-2000
Intestine: • Desmin (m), VECTASTAIN® Elite ® ABC Kit, ImmPACT™ DAB (brown) Tonsil: • Cyclin A (m), VECTASTAIN® Universal ABC-AP Kit, Vector® Blue AP Substrate Anti-Mouse IgG (H+L) made in horse, rat adsorbed, biotinylated BA-2001
substrate. • Cytokeratin (m), VECTASTAIN® Elite ® ABC Kit, Vector® VIP (purple) (blue) • CD20 (m), VECTASTAIN® Universal Elite ® ABC Kit, Vector® NovaRED™ HRP Anti-Mouse IgG (H+L) made in goat, biotinylated BA-9200 BP-9200
substrate. Substrate (red).
Anti-Mouse IgM (H+L) μ chain specific, made in goat, biotinylated BA-2020
Anti-Rabbit IgG (H+L) made in goat, biotinylated BA-1000 BP-9100
Anti-Rabbit IgG (H+L) made in horse, biotinylated BA-1100 BP-1100
Anti-Rat IgG (H+L) made in rabbit, biotinylated BA-4000
Anti-Rat IgG (H+L) made in rabbit, mouse adsorbed, biotinylated BA-4001
Anti-Rat IgG (H+L) made in goat, biotinylated BA-9400 BP-9400
Anti-Rat IgG (H+L) made in goat, mouse adsorbed, biotinylated BA-9401
Universal Anti-Mouse/Rabbit IgG (H+L) made in horse, biotinylated BA-1400 BP-1400
Universal Pan-Specific Anti-Mouse/Rabbit/Goat IgG (H+L) made in horse, biotinylated BA-1300

3. Choose the blocking solution

Product Concentrate R.T.U.†

Normal Goat Serum S-1000 S-1012
Normal Rabbit Serum S-5000
Normal Horse Serum S-2000 S-2012
Breast Carcinoma: • Estrogen Receptor (m); VECTASTAIN® Elite ® ABC Kit, Vector® Tonsil: • CD3 (m), VECTASTAIN® Universal Elite ® ABC Kit, Vector® VIP substrate Animal-Free Block and Diluent SP-5030 SP-5035
NovaRED substrate (red) • CD34 (m), VECTASTAIN® Elite ® ABC Kit, DAB substrate (purple) • CD20 (m), VECTASTAIN® Universal Elite ® ABC Kit, Vector® SG substrate
(brown) • Cytokeratin 8/18 (m), VECTASTAIN® Elite ® ABC Kit, Vector® SG substrate (blue/gray). *
For a complete list of all biotinylated secondary antibodies please visit: https://vectorlabs.com/b-2nd-abs/
(blue/gray). †
Ready-to-use, prediluted stabilized solutions.

16 vectorlabs.com vectorlabs.com 17
 Choosing a Detection System

Polymer-Based Detection
Non-biotin micropolymer-based detection for greater signal, low background,
and superior access to epitopes
Polymer-based reagents are a more recent introduction into IHC detection methodology than traditional avidin and biotin
conjugates, such as ABC kit formats. Polymers offer distinct advantages over these traditional methods particularly for
applications such as multiple antigen labeling (multiplexing) on the same tissue section, or in instances where detectable
levels of endogenous biotin may be problematic.
Polymer-based systems essentially consist of an integrated polymer of active enzyme and secondary antibody that binds to
a primary antibody target. This integrated format introduces significantly more enzyme at the site of localization, thereby
generating a greater reaction with the subsequent chromogen, compared with a secondary antibody directly conjugated with
enzyme. Additionally, use of a one-step polymer method shortens the IHC procedure by avoiding the two-step biotinylated
secondary antibody and ABC reagent that are required for standard avidin-biotin systems.
Polymer-based systems were initially introduced consisting of dextran or similar molecules that had inherent issues in some Breast Carcinoma: • Estrogen Receptor (m), ImmPRESS™ Reagent (HRP; Breast Carcinoma: • Estrogen Receptor (m), ImmPRESS™ Reagent (HRP;
tissues due to their large size. Our ImmPRESS™ polymer systems have been highly refined and consist of micropolymers that Universal), Vector® DAB (brown) • Cytokeratin AE1/AE3 (m), VECTASTAIN® ABC-AP Universal), Vector® NovaRED™ HRP Substrate (red) • CD34 (m), ImmPRESS™
penetrate more easily into thicker sections, avoid steric hindrance concerns, and provide defined, specific binding to the Kit (Universal), Vector® Red (magenta). Reagent (HRP; Universal), Vector® DAB+Ni HRP Substrate (gray-black).
primary antibody.

Small Bowel: • Neurofilament 200 kDa (m), ImmPRESS™ Reagent (HRP) Breast Carcinoma: • Estrogen Receptor (m), ImmPRESS™ Reagent (HRP;
Anti-Mouse IgG, Vector® VIP (purple) • Desmin (m), ImmPRESS™ Reagent (HRP) Universal), Vector® NovaRED™ (red) • CD34 (m), VECTASTAIN® ABC-AP Kit
Anti-Mouse IgG, Vector® SG (blue-gray). (Universal), Vector® Blue (blue).

Mouse Tonsil: CD45 (rat), ImmPRESS™ Reagent (HRP) Anti-Rat IgG, Mouse Small Bowel: • CD10 (m), ImmPRESS™ Reagent (HRP) Anti-Mouse IgG, Vector®
Adsorbed, ImmPACT™ AEC (red), Hematoxylin QS (blue). DAB-Ni (black) • Cytokeratin 20 (m), ImmPRESS™ Reagent (HRP) Anti-Mouse IgG,
Small Bowel: • Ki67 (rp), ImmPRESS™ Reagent (HRP; Universal), Vector® NovaRED™ (red) • Cytokeratin 8/18 (m), ImmPRESS™ Reagent (HRP; Universal), Vector® SG (blue-gray). Vector® NovaRED™ (red).

18 vectorlabs.com vectorlabs.com 19
 Polymer-Based Detection

ImmPRESS™ One-Step Polymer Systems ImmPRESS™ Two-Step Amplified Polymer Systems

ImmPRESS™ Polymer Detection Systems ImmPRESS™ Polymer Kits
ImmPRESS™ Excel Amplified Polymer Staining Systems ImmPRESS™ Excel Amplified
ImmPRESS™ Polymer Reagents consist of unique micropolymers of highly active (Peroxidase) ImmPRESS™ Excel Amplified Peroxidase (HRP) Polymer Staining Systems are complete Polymer Kits (Peroxidase)
peroxidase or alkaline phosphatase enzyme attached to highly cross-adsorbed, affinity- staining kits that capitalize on all the advantages of the ImmPRESS™ HRP Polymer System
purified secondary antibodies. This micropolymer conjugation technology allows a higher Anti-Rabbit IgG technology and offer additional sensitivity and convenience. Anti-Rabbit IgG
density of enzymes per antibody to bind to the target with minimal steric interference. Anti-Mouse IgG Anti-Mouse IgG
The ImmPRESS™ Polymer Reagents produce outstanding immunohistochemistry and This system employs a ready-to-use (R.T.U.) Amplifier Antibody, followed by an
Anti-Mouse IgG, Rat Adsorbed ImmPRESS™ Excel HRP Polymer Reagent. These reagents are affinity-purified and
immunocytochemistry results due to increased target accessibility, binding specificity, and
signal intensity along with low background staining. Anti-Rat IgG extensively cross-adsorbed to ensure high sensitivity and low background. The included
ImmPACT™ DAB EqV (equal volume) substrate produces a crisp, dark brown reaction
• High sensitivity and very low background for crisp, strong staining Anti-Rat IgG, Mouse Adsorbed
product with excellent sensitivity that is characteristic of the ImmPRESS™/ImmPACT™
• Ready-to-use, one-step detection system – no mixing or titering Anti-Goat IgG combination.
• Includes prediluted blocking serum Universal Antibody,
The ImmPRESS™ Excel Amplified HRP Kits are recommended for applications that
Anti-Rabbit/Mouse IgG
• Shorter assay time require detection of weakly-expressed antigens, in cases of unknown expression levels
• Non-biotin based such as gene knock-in studies, or in determining up-regulation of a given target. This
straightforward kit format yields reliable, consistent results and saves time in trying to
• Excellent resolution ImmPRESS™ Polymer Kits establish optimal titrations with concentrated detection reagents.
• Especially suited for nuclear and membrane antigens (Alkaline Phosphatase)
Each ImmPRESS™ Excel Amplified Staining Kit includes the following:
• Ideal for multiple antigen labeling (p. 30-33)
Anti-Rabbit IgG • BLOXALL™ Endogenous Enzyme Blocking Solution
Anti-Mouse IgG • 2.5% Normal Horse Serum
ImmPRESS™ VR Polymer Kits Anti-Rat IgG • Amplifier Antibody (goat anti-rabbit IgG or goat anti-mouse IgG)
ImmPRESS™ VR (Veterinary Reagents) Kits are available additionally cross-adsorbed to
Anti-Rat IgG, Mouse Adsorbed • ImmPRESS™ Excel Polymer Detection Reagent (horse anti-goat IgG)
ensure minimal cross-reactivity against endogenous tissue elements in animal species
commonly used for diagnostics and research-based animal models (bovine, goat, sheep, Anti-Goat IgG • ImmPACT™ DAB EqV Substrate (Chromogen and Buffer)
swine, horse, cat, dog, rabbit, rat, mouse).
Using the ImmPRESS™ Two-Step Amplified Polymer Systems
ImmPRESS™ VR Polymer Kits
(Peroxidase)
1 Add Primary Antibody 2 Add Amplifier Antibody
Anti-Rabbit IgG
Anti-Mouse IgG

Using the ImmPRESS™ Polymer Kits

1 Add Primary Antibody 2 Add ImmPRESS™ Reagent 3 Add Enzyme Substrate

3 Add ImmPRESS™ Excel HRP 4 Add Enzyme Substrate

Polymer Reagent

20 vectorlabs.com vectorlabs.com 21
 Polymer-Based Detection

Choosing an ImmPRESS™ Polymer Kit Multiple Antigen Labeling Simplified

When choosing the optimal detection system for your application, consider not A key advantage of the ImmPRESS™ Polymer Reagent is that it significantly shortens staining times for multiple antigen
only the species of the primary antibody but also the species of the tissue under labeling (multiplexing).
examination. If the species of the primary antibody and the species of the tissue are • Fewer steps than conventional protocols decreases slide handling.
closely related (for example, rat and mouse), the secondary antibody may cross-react
with endogenous IgG in the tissue section. This can lead to background staining. For • Eliminates the need for avidin/biotin blocking steps in samples with endogenous biotin.
the two-step ImmPRESS™ Excel Amplified Polymer Staining Kit, the species of the
Amplifier Antibody (made in goat) should be considered in addition to the primary
and tissue species.
The following options can minimize background staining in these instances:
• Use a secondary antibody specifically adsorbed to remove cross-reacting antibodies
of closely-related species (for example, ImmPRESS™ Anti-Mouse IgG, Rat Adsorbed).
• Use the ImmPRESS™ M.O.M.™ Kit for applications of mouse primary antibodies on
mouse tissue. (p. 25)
• Use ImmPRESS™ VR Polymer Detection Reagents, which eliminate off-target binding
and thus increase signal specificity in these animal tissues: bovine, goat, sheep,
swine, horse, cat, dog, rabbit, rat, mouse.
Breast Carcinoma: • Ki67 (rm), ImmPRESS™ Reagent Colon: • M2A antigen (m), VECTASTAIN® ABC-AP Colon: • CD3 (rm), ImmPRESS™ Reagent (HRP)
• Since the Amplifier Antibody in both the ImmPRESS™ Excel Anti-Mouse IgG (HRP; Universal), Vector® DAB (brown) • CD34, Kit (Universal), Vector® Blue (blue) • CD20 (m), Anti-Rabbit IgG, ImmPACT™ AMEC Red • CD34 (m),
Kit and the ImmPRESS™ Excel Anti-Rabbit Kit is made in goat, these kits are ImmPRESS™ Reagent (HRP; Universal), Vector® ImmPRESS™ Reagent (HRP; Universal), Vector® ImmPRESS™-AP Anti-Mouse IgG Reagent, Vector® Blue
VIP (purple). VIP (purple). (blue).
not recommended for goat, bovine and sheep tissue - the ImmPRESS™ Excel
Polymer Detection Reagent (an anti-goat IgG) may cross-react with endogenous
immunoglobulins.
Using the ImmPRESS™ Polymer Kits for multiple antigen labeling
1 Add First Primary Antibody 2 Add ImmPRESS™ Reagent 3 Add Enzyme Substrate I

Lung: aSMA (m), ImmPRESS™-AP Anti-Mouse IgG, Canine small intestine: Cytokeratin (MNF 116),
ImmPACT™ Vector® Red AP Substrate. Counterstained ImmPRESS™ VR Anti-Mouse IgG and Vector® DAB Sub-
with Hematoxylin (blue). Image kindly provided by the strate (brown). Counterstained with hematoxylin (blue).
Centre for Inflammation and Tissue Repair, University
College London.
4 Add Second Primary Antibody 5 Add ImmPRESS™ Reagent 6 Add Enzyme Substrate II
Veterinary Excel Amplified Alkaline
Product Peroxidase Reagents (VR) (Peroxidase) Phosphatase
ImmPRESS™ Anti-Rabbit IgG Kit (made in horse) MP-7401 MP-6401 MP-7601 MP-5401
ImmPRESS™ Anti-Rabbit IgG Kit (made in goat) MP-7451
ImmPRESS™ Anti-Mouse IgG Kit (made in horse) MP-7402 MP-6402 MP-7602 MP-5402
ImmPRESS™ Anti-Mouse IgG Kit (made in goat) MP-7452
ImmPRESS™ Anti-Mouse IgG, Rat Adsorbed, Kit (made in horse) MP-7422
ImmPRESS™ Anti-Rat IgG Kit (made in horse) MP-7404 MP-5404
ImmPRESS™ Anti-Rat IgG, Mouse Adsorbed, Kit (made in horse) MP-7444 MP-5444
ImmPRESS™ Anti-Goat IgG Kit (made in horse) MP-7405 MP-5405
ImmPRESS™ Universal Antibody Kit, Anti-Rabbit/Mouse Kit (made in horse) MP-7500 Read more: Discovery Through Color: A Guide to Multiple Antigen Labeling (vectorlabs.com/brochures)

22 vectorlabs.com vectorlabs.com 23
 Species on Species Detection Mouse on Mouse (M.O.M.™) Immunodetection Kits
(Mouse)
Vector Laboratories M.O.M.™ Immunodetection systems are specifically designed to
Recommended applications:
localize mouse primary antibodies on mouse tissue while avoiding background staining.
These M.O.M.™ Kits contain our proprietary M.O.M.™ Mouse Ig Blocking Reagent.
Solutions when your primary antibody is the same species M.O.M.™ Kits are available based on either avidin-biotin technology (M.O.M.™ Elite®
Studies in genetically
engineered mice
as your specimen. ABC Kit, Fluorescein Kit, or Basic Kit) or polymer technology (M.O.M.™ ImmPRESS™ HRP
Polymer Kit). Use the M.O.M.™ Immunodetection systems to introduce two or more Transgenic and knock-out models
When a primary antibody is the same species as the specimen, the
secondary antibody cannot distinguish between the endogenous different labels using a multiple antigen labeling protocol. You can detect several mouse Mouse xenograft tissue
primary antibodies on the same tissue section, regardless of the species of the tissue. Normal mouse tissue
immunoglobulins and the primary antibody. This can result in high Excellent staining results for a once difficult application have now become routine with
background staining that obscures antigen-specific staining. Mouse on the Vector M.O.M.™ System.
Mouse detection is especially important because of the vast number of
• Significantly reduces endogenous mouse Ig staining when using mouse primary
primary antibodies made in mouse and the wide use of mice in model
antibodies on mouse tissue
systems, xenografts, and other applications.
• Simple protocols
• Eliminates tedious calculations
Newborn Mouse Tongue: • Synapsin (m), M.O.M.™ Peroxidase Kit, Vector® NovaRED™ (red) • Desmin (m), M.O.M.™ • Eliminates primary antibody prebinding steps
Peroxidase Kit, Vector® DAB-Ni (black).
• Clear, crisp, specific staining of antigens of interest
• Compatible with fluorescent or enzyme-based detection Sections of mouse kidney stained with
mouse antibody against smooth muscle
• Available with or without enzyme or fluorochrome actin using VECTASTAIN® ABC-AP Kit and
Vector® Blue substrate. Using standard
biotinylated anti-mouse antibody and
normal blocking serum, confusing
background is seen (left). With the Vector®
M.O.M.™ Basic Kit, clean background and
specific staining is achieved (right).

Mouse intestine stained with standard anti-mouse IgG polymer system (left) and Vector® M.O.M.™ ImmPRESS™
HRP Polymer Kit (right). Brown signal indicates IgG background staining. (Both: No primary antibody, Vector® DAB
stain, hematoxylin counterstain.)

Product Catalog Number

M.O.M.™ Peroxidase Kit PK-2200
M.O.M.™ Fluorescein Kit FMK-2201
M.O.M.™ Basic Kit BMK-2202
M.O.M.™ ImmPRESS™ HRP Polymer Kit MP-2400
M.O.M.™ Mouse Ig Blocking Reagent MKB-2213
M.O.M.™ Biotinylated Anti-Mouse Ig Reagent* MKB-2225
M.O.M.™ ImmPRESS™ HRP Polymer Anti-Mouse Reagent MPX-2402

* This reagent must be used with the M.O.M.™ Mouse Ig Blocking Reagent (MKB-2213). It is not intended to be a
stand-alone reagent for mouse on mouse applications.

24 vectorlabs.com vectorlabs.com 25
 Choosing an Enzyme Substrate Enzyme Substrates
Vector Laboratories enzyme substrates produce a range of We offer researchers an array of both conventional and unique enzyme substrates that produce a broad range of colors.
Our reagents require no dissolving of powders or tablets and are provided in convenient dropper bottles which
sensitivities across a broad palette of colors.
are safe and easy to handle.
Consider the following factors when choosing a substrate to match the
enzyme in your detection system and your application.
• Sensitivity. Substrates differ in sensitivity. Some may increase in
sensitivity with longer incubation times.
Relative sensitivity of substrates in IHC
• Color. Color contrast is essential in multiple antigen labeling Lower Sensitivity Higher Sensitivity
applications, in pigmented tissues such as melanomas, and in
counterstained tissues. Where performance is equal, color choices Vector® SG ImmPACT™
SG
Vector® DAB
(with Nickel)

might also depend on personal preference.

• Visualization. Visualization options include brightfield, fluorescence, Peroxidase (HRP)
Competitor
DAB tablets
Vector® DAB ImmPACT™
DAB
darkfield, electron microscopy, and spectral imaging. HRP substrates ImmPACT™
were used with the DAB EqV
• Heat Resistance. For IHC/ISH double-labeling applications, the heat- ImmPRESS™ (HRP)
ImmPACT™
resistant substrate is applied first with an IHC protocol, followed by ISH Polymer Detection Vector® VIP
VIP
Reagent.
detection that includes a heat denaturation step. In multiple antigen TMB
labeling procedures requiring additional applications of heat-induced *

epitope retrieval (HIER), apply the heat-resistant substrate first. Vector®

NovaRED™
ImmPACT™
NovaRED™

Tonsil: Cytokeratin AE1/AE3 (m), ImmPRESS™-AP Anti-Mouse IgG, Vector® Blue (blue). ImmPACT™ ImmPACT™
Vector® AEC AMEC Red
AEC

Vector® Red ImmPACT®

Alkaline Vector® Red
Phosphatase (AP)
AP substrates were Vector®
Blue
used with the
ImmPRESS™ (AP)
BCIP/NBT
Polymer Detection **
Reagent.

Vector®
Black

* Reaction product deposition not discreet and can be variable

** Longer incubation times increase sensitivity

26 vectorlabs.com vectorlabs.com 27
 Choosing an Enzyme Substrate

Enzyme Substrate Properties

See also Enzyme Substrate Combinations for multiple antigen labeling (p. 30-33) and Counterstain/Substrate Compatibility (p. 35). Peroxidase Substrates Alkaline Phosphatase Substrates

Microscopy
Contrast in
Catalog Bright- Fluore- Spectral Pigmented Multiple Heat
Substrate Color Number field Darkfield Electron scence Imaging Mounting Tissue Labeling Resistant*
Peroxidase
Vector® DAB Brown SK-4100 • • • • Non-aqueous • •
or Aqueous
Vector® DAB +Ni Gray-Black SK-4100 • • • • Non-aqueous •
ImmPACT™ DAB Brown SK-4105 • • • • Non-aqueous • • Prostate: Prostate Specific Antigen (m), ImmPRESS™ Tonsil: Cytokeratin AE1/AE3 (m), ImmPRESS™ Tonsil: Cytokeratin AE1/AE3 (m), Vector® ImmPRESS™-
or Aqueous
Reagent (HRP), ImmPACT™ DAB (brown). Reagent (HRP), Vector® DAB-Ni (gray-black). AP Reagent, ImmPACT™ Vector® Red (magenta).
ImmPACT™ Brown SK-4103 • • • • Non-aqueous • •
DAB EqV or Aqueous
Vector® VIP Purple SK-4600 • • • • Non-aqueous • •
ImmPACT™ VIP Purple SK-4605 • • • • Non-aqueous • •
Vector® SG Blue-Gray SK-4700 • • • • Non-aqueous • •
or Aqueous
ImmPACT™ SG Blue-Gray SK-4705 • • • • Non-aqueous • •
or Aqueous
Vector® NovaRED™ Red SK-4800 • • • • Non-aqueous • •
Tonsil: Cytokeratin AE1/AE3 (m), ImmPRESS™ Tumor: Cytokeratin (s), VECTASTAIN® Elite® ABC Kit, Tonsil: Cytokeratin AE1/AE3 (m), ImmPRESS™-AP
ImmPACT™ Red SK-4805 • • • • Non-aqueous • • Reagent (HRP), ImmPACT™ SG (blue-gray). TMB (blue). Reagent, Vector® Blue (blue).
NovaRED™
Vector® AEC Red SK-4200 • • Aqueous • •
ImmPACT™ AEC Red SK-4205 • • Aqueous • •
ImmPACT™ Red SK-4285 • • Aqueous • •
AMEC Red
TMB Blue SK-4400 • • Non-aqueous

Alkaline Phosphatase
Vector® Red Magenta SK-5100 • • • Non-aqueous • • •
or Aqueous
Tonsil: Cytokeratin AE1/AE3 (m), ImmPRESS™ Prostate: Prostate Specific Antigen (m), ImmPRESS™ Colon Carcinoma: Pan-Cytokeratin (m), VECTASTAIN®
ImmPACT™ Magenta SK-5105 • • • Non-aqueous • • • Reagent (HRP), ImmPACT™ NovaRED™ (red). Reagent (HRP), ImmPACT™ AEC (red). ABC-AP Kit, Vector® Black (brown-black).
Vector® Red or Aqueous

Vector® Blue Blue SK-5300 • • • Non-aqueous • • •

or Aqueous
Vector® Black Brown-Black SK-5200 • Non-aqueous

BCIP/NBT Indigo SK-5400 • • • Non-aqueous • •

or Aqueous

* Substrates that are designated “heat resistant” were developed on tissue then subjected to heat induced epitope retrival (HIER) using a pressure cooker technique (stained
tissue was pressure cooked for 1 minute in Antigen Unmasking Solution, returned to room temperature, and rinsed in buffer). Resulting sensitivity after this treatment was
found to be equivalent to non-HIER treated tissue.

Tonsil: CD20 (m), ImmPRESS™ Reagent (HRP), Tonsil: LCA (m), ImmPRESS™ Reagent (HRP), Prostate: Prostate Specific Antigen (m), VECTASTAIN®
ImmPACT™ VIP (purple). ImmPACT™ AMEC Red (red). ABC-AP Kit, BCIP/NBT (indigo).

28 vectorlabs.com vectorlabs.com 29
 Multiple Antigen Labeling Enzyme Substrate Combinations
Localization of two or more antigens on the same tissue section is a powerful Recommended combinations of substrates and the recommended order in which they should be used.
research tool that can provide valuable insights into cellular biochemistry,
Second Substrate ImmPACT™ Vector® Blue BCIP/NBT ImmPACT™ ImmPACT™ DAB, DAB-Ni ImmPACT™ ImmPACT™ ImmPACT™ ImmPACT™
protein-protein interactions, and spatial relationships of biomarkers. Vector® Red & (blue) (indigo) VIP & mmPACT™ DAB (gray-black) NovaRED™ & SG & AEC & AMEC Red
Vector® Red SK-5300 SK-5400 Vector® VIP EqV & Vector® SK-4100 Vector® Vector® SG Vector® (red)
Our detection systems and enzyme substrates have been developed and (magenta) (purple) DAB (brown) NovaRED™ (red) (blue-gray) AEC (red) SK-4285
rigorously tested to deliver the high sensitivity, low background, and extreme SK-5105 & SK-4605 & SK-4105, SK-4805 & SK-4705 & SK-4205 &
First Substrate SK-5100 SK-4600 SK-4103, SK-4100 SK-4800 SK-4700 SK-4200
clarity that is required to differentiate multiple epitopes simultaneously.
ImmPACT™ Vector® Red
You can choose to use the same enzyme system with different substrates or & Vector® Red (magenta)
different enzyme systems and their respective substrates. Cat. No. SK-5105 & – – – + + – + – –
SK-5100
For a detailed description of these applications, protocols, and additional Vector® Blue (blue)
images please visit our website or request a free copy of our guide, Cat. No. SK-5300 + – + + + + + + +
Discovery Through Color: A Guide to Multiple Antigen Labeling. BCIP/NBT (indigo)
+ – + + + + + + +
Cat. No. SK-5400
ImmPACT™ VIP &
Colon: • Cytokeratin (AE1/AE3, m), ImmPRESS™-AP Anti-Mouse IgG Reagent, Vector® Blue AP Substrate (blue)
• CD3 (rb), ImmPRESS™ Anti-Rabbit IgG HRP Reagent, ImmPACT™ AMEC Red HRP Substrate (red).
Vector® VIP (purple) – + – + + – + – –
Cat. No. SK-4605, SK-4600
ImmPACT™ DAB,
ImmPACT™ DAB EqV &
Vector® DAB (brown) + + + + – – + + +
Cat. No. SK-4105,
SK-4103, SK-4100
Vector® DAB-Ni
(gray-black) + – – + + + – – –
Cat. No. SK-4100
ImmPACT™ NovaRED™
& Vector® NovaRED™
(red) – + + – + + + – –
Cat. No. SK-4805, SK-4800
ImmPACT™ SG &
Vector® SG (blue-gray) + – – + + – – + +
Cat. No. SK-4705, SK-4700
ImmPACT™ AEC &
Vector® AEC (red) – – – – + – – + –
Cat. No. SK-4205, SK-4200
ImmPACT™ AMEC Red
(red) – – – – + – – + –
SK-4285

Key: Alkaline Phosphatase Peroxidase + Indicates good contrast – Indicates incompatiblity of substrates for various reasons

Breast Carcinoma: • Estrogen Receptor (m), VECTASTAIN®
Elite® ABC Kit, Vector® NovaRED™ HRP substrate (red)
• CD34 (m), VECTASTAIN® ABC-AP Kit, Vector® Blue AP
Substrate (blue) • Cytokeratin 8/18 (m), VECTASTAIN®
Elite® ABC Kit, Vector®DAB HRP Substrate (brown).
Tonsil: • CD3 (m), ImmPRESS™ Anti-Mouse IgG Prostate: • Cytokeratin 5 (m), VECTASTAIN® Universal
Reagent, Vector® VIP HRP Substrate (purple) • Ki67 (m), ABC-AP Kit, Vector® Blue AP Substrate (blue) • CD34
ImmPRESS™ Anti-Mouse IgG Reagent, Vector® SG HRP (m), VECTASTAIN® Universal ABC-AP Kit, Vector® Red
Substrate (blue/gray). AP Substrate (red).

30 vectorlabs.com vectorlabs.com 31
 Multiple Antigen Labeling

Multiple Labeling Examples

Colon Cancer: • Ki67 (rm), ImmPRESS™-AP Anti-Rabbit IgG Reagent, Vector® Blue AP Colon: • Ki67 (rm), ImmPRESS™ Universal (Anti-Mouse/Anti-Rabbit IgG) HRP Tonsil: • CD3 (m), VECTASTAIN® Elite® ABC Kit (Universal), Vector® DAB (brown) Breast Carcinoma: • Estrogen Receptor (rm), ImmPRESS™ Universal Reagent,
Substrate (blue) • Cox2 (rm), ImmPRESS™-AP Anti-Rabbit IgG Reagent, ImmPACT™ Reagent, Vector® VIP (purple) • Cytokeratin (m), ImmPRESS™ Universal (Anti- • CD20 (m), VECTASTAIN® Elite® ABC Kit (Universal), Vector® SG (blue-gray) Vector® NovaRED™ HRP Substrate (red) • M2A Antigen (m), ImmPRESS™
Vector® Red AP Substrate (red). Mouse/Anti-Rabbit IgG) HRP Reagent, Vector® SG HRP Substrate (gray). • Multi-Cytokeratin (m), VECTASTAIN® Elite® ABC Kit (Universal), Vector® VIP Universal HRP Reagent, Vector® DAB+Ni HRP Substrate (gray/black).
(purple).

Breast Carcinoma: • Ki67 (rm), ImmPRESS™ Reagent (HRP; Universal), Vector® DAB Melanoma: • Cyclin A (m), ImmPRESS™-AP Anti-Mouse IgG, ImmPACT™ Vector® Colon: • M2A Antigen (m), ImmPRESS™ Universal (Anti-Mouse/Rabbit IgG) Colon Carcinoma: • S100 (rp), VECTASTAIN® Universal ABC-AP Kit, Vector® Red
(brown) • CD34 (m), ImmPRESS™ Reagent (HRP; Universal), Vector® VIP (purple). Red AP Substrate (magenta) • Melanoma Marker (m) ImmPRESS™ HRP Anti- HRP Reagent, Vector® NovaRED HRP Substrate (red) • CD34 (m), VECTASTAIN® AP Substrate (red) • Cytokeratin 8/18 (m), VECTASTAIN® Universal Elite® ABC Kit,
Mouse IgG, Vector® SG HRP Substrate (gray). Note contrast of double stain with Universal ABC-AP kit, Vector® Blue AP Substrate (blue). Vector® SG HRP Substrate (blue/gray).
the brown pigments in the tissue.

32 vectorlabs.com vectorlabs.com 33
 Counterstaining
A counterstain introduces color to specific cellular structures to provide contrast to the colored enzyme substrate.
Counterstaining aids in visualization and target localization, facilitating interpretation of morphology and cell structure within
the tissue section. Our nuclear counterstains are packaged as convenient, ready-to-use solutions for use on individual slides or in
staining dishes.
Counterstain/Substrate Compatibility Table
This table is designed as a reference to determine the optimal counterstain/substrate combination for your application.
Considerations should be given to tissue type, antigen unmasking protocol and other detection parameters to achieve
the desired staining intensity.

Vector® Hematoxylin Vector® Vector®

Vector® Hematoxylin (blue) Catalog and Hematoxylin QS Methyl Green Nuclear Fast Red
• Based on Gill’s III formulation Substrate Number H-3401 and H-3404 H-3402 H-3403
• Progressive stain formula. The intensity can be adjusted to ImmPACT™ DAB (brown) SK-4105 Excellent Contrast Excellent Contrast Fair Contrast
optimize results for either manual or automated systems
ImmPACT™ DAB EqV SK-4103 Excellent Contrast Excellent Contrast Fair Contrast
• Excellent color contrast with most commonly used
Vector® DAB (brown) SK-4100 Excellent Contrast Excellent Contrast Fair Contrast
peroxidase and alkaline phosphatase substrates
• Suitable for use with non-aqueous and aqueous Vector® DAB-Ni (gray-black) SK-4100 Excellent Contrast Fair Contrast * Good Contrast
mounting media ImmPACT™ AEC (red) SK-4205 Excellent Contrast Counterstain Incompatibility ** Color Incompatibility
• Alcohol- and mercury-free ImmPACT™ AMEC Red (red) SK-4285 Excellent Contrast Counterstain Incompatibility ** Color Incompatibility

Vector® AEC (red) SK-4200 Excellent Contrast Counterstain Incompatibility ** Color Incompatibility
Tumor tissue section showing specific cytoplasmic cell staining (Vector® DAB,
Vector® Hematoxylin QS (blue) brown) with Vector® Hematoxylin QS counterstain (blue). TMB (blue) SK-4400 Color Incompatibility Counterstain Incompatibility Excellent Contrast
• Modification of Mayer’s hematoxylin developed especially
ImmPACT™ VIP (purple) SK-4605 Fair Contrast Excellent Contrast Poor Contrast
for immunocytochemistry
Vector® VIP (purple) SK-4600 Fair Contrast Excellent Contrast Poor Contrast
• Ready-to-use without filtration or ‘blueing’ step
• Stains in less than 45 seconds ImmPACT™ SG (blue-gray) SK-4705 Poor Contrast Good Contrast Excellent Contrast

• Excellent color contrast with most commonly used Vector® SG (blue-gray) SK-4700 Poor Contrast Good Contrast Excellent Contrast
peroxidase and alkaline phosphatase substrates ImmPACT™ NovaRED™ (red) SK-4805 Excellent Contrast Excellent Contrast *** Color Incompatibility
• Suitable for use with non-aqueous and aqueous Vector® NovaRED™ (red) SK-4800 Excellent Contrast Excellent Contrast *** Color Incompatibility
mounting media
ImmPACT™ Vector® Red (magenta) SK-5105 Excellent Contrast Excellent Contrast Color Incompatibility
• Mercury-free
Vector® Red (magenta) SK-5100 Excellent Contrast Excellent Contrast Color Incompatibility

Vector® Black (black) SK-5200 Excellent Contrast Excellent Contrast * Excellent Contrast
Vector® Methyl Green (light green)
Tonsil section showing specific cell staining (Vector® VIP, purple) with Vector® Vector® Blue (blue) SK-5300 Color Incompatibility Good Contrast Excellent Contrast
• Superior formulation of methyl green suitable for use with Methyl Green counterstain (green).
a wide range of enzyme substrates BCIP/NBT (indigo) SK-5400 Color Incompatibility Excellent Contrast * Excellent Contrast
• Simple, two-step procedure
* This substrate shows a slight decrease in sensitivity following the methyl green protocol. This decrease can be minimized by reducing the heat incubation
• Excellent alternative in multiple antigen labeling when and acetone rinse times in the methyl green protocol.
hematoxylin obscures the substrate colors ** Substrate dissolves in acetone wash.
*** A slight color change in ImmPACT™ NovaRED™ and Vector® NovaRED™ reaction product may be seen using methyl green.
• Suitable for use with non-aqueous mounting media

Vector® Nuclear Fast Red (pink) Product Mountants Catalog Number

• Fast one-step protocol Vector® Hematoxylin Non-aqueous and Aqueous H-3401
• Excellent alternative in multiple antigen labeling when Vector® Hematoxylin QS Non-aqueous and Aqueous H-3404
hematoxylin obscures the substrate colors
Vector® Methyl Green Non-aqueous H-3402
• Good contrast with a variety of substrates
Tumor tissue section showing specific cytoplasmic cell staining (Vector® SG, blue/ Vector® Nuclear Fast Red Non-aqueous and Aqueous H-3403
gray) with Vector® Nuclear Fast Red counterstain (red).

34 vectorlabs.com vectorlabs.com 35
 Blocking Background Signal
Blocking agents minimize background signal from endogenous enzyme activity, biotin, and non-specific binding of tissue 2.5% Normal Sera R.T.U. Animal-Free Block and Diluent is a unique formulation
elements (charged particles, macromolecules, Fc receptors) with detection reagents. Our 2.5% Normal Sera are pooled samples collected from different from our concentrated (5x) animal-free blocker. It
healthy adult animals. has been designed with optimized conditions and neutral pH
specifically for IHC and IF methods.
• Heat-treated and centrifuged to remove precipitates and
then filtered
• Tested for cross-reactivities Animal-Free Blocker™ (5x concentrate solution)
• Can be used for blocking non-specific binding or as an Animal-Free Blocker™ is a plant-derived blocking agent and
antibody diluent diluent for IHC, nucleic acid blotting, protein blotting, and
other applications. This reagent contains no animal-derived
protein and can be used as an alternative to sera, BSA, casein,
Bovine Serum Albumin (BSA) or non-fat dry milk.
Immunohistochemical Grade.
• Can be used as a diluent or a blocking agent
• Free of impurities present in other grades of BSA, which Product Catalog Number
can introduce artifacts or increase background staining in BLOXALL™ Blocking Solution SP-6000
IHC staining, ELISAs, or blots
Levamisole Solution SP-5000

10x Casein Solution Avidin/Biotin Blocking Kit SP-2001

Endogenous alkaline phosphatase (AP) and peroxidase (HRP) activities in frozen, acetone-fixed intestine revealed with Vector® Red AP Substrate (magenta) and
ImmPACT™ DAB HRP Substrate (brown), (left). Same substrates used on BLOXALL™ Solution-treated tissue (right). BLOXALL™ Blocking Solution completely eliminates 10x Casein Solution is a general blocking agent for IHC, nucleic
both endogenous enzyme activities. acid blotting, protein blotting, and other applications. Streptavidin/Biotin Blocking Kit SP-2002

Normal Goat Serum S-1000

BLOXALL™ Endogenous Peroxidase/Alkaline Avidin/Biotin Blocking Kit Carbo-Free™ Blocking Solution
Phosphatase Blocking Solution The Avidin/Biotin Blocking Kit blocks all endogenous biotin, Normal Horse Serum S-2000
Carbo-Free Blocking Solution is a protein-based agent that
Tissues may contain endogenous peroxidase, biotin receptors, and avidin binding sites present in tissues to is essentially free of glycoproteins. It is ideal for applications Normal Chicken Serum S-3000
pseudoperoxidase, and/or alkaline phosphatase activity that prevent non-specific binding of avidin or biotinylated reagents using lectins in which glycoprotein contamination could
will produce background staining. BLOXALL™ Endogenous with avidin-biotin detection systems. Ready-to-use in a generate background staining or false positive results. Normal Swine Serum S-4000
Peroxidase/Alkaline Phosphatase Blocking Solution inactivates convenient dropper bottle.
• Can be used to block non-specific binding or as an
each of these enzymes in one step. Normal Rabbit Serum S-5000
antibody diluent
• Compatible with formalin-fixed, paraffin-embedded tissue Streptavidin/Biotin Blocking Kit 2.5% Normal Goat Serum S-1012
sections, frozen tissue sections, and cell preparations
Streptavidin/Biotin Blocking Kit blocks all endogenous biotin, R.T.U. Animal-Free Block and Diluent
• Ready-to-use in a convenient dropper bottle 2.5% Normal Horse Serum S-2012
biotin receptors, and streptavidin binding sites present in
This plant protein-derived product is a universal antibody
• More effective than conventional blocking methods tissues to prevent non-specific binding of streptavidin or Bovine Serum Albumin (BSA) SP-5050
diluent and blocking reagent intended for cell- and tissue-
• Brief 10-minute incubation biotinylated reagents with biotin/streptavidin detection
based IHC and IF applications. This ready-to-use solution can
systems. Ready-to-use in a convenient dropper bottle. 10x Casein Solution SP-5020
be used as an alternative to normal sera, BSA, casein and
Levamisole Solution non-fat dry milk. Carbo-Free™ Blocking Solution SP-5040
Normal Sera
Levamisole Solution specifically inhibits endogenous alkaline R.T.U. Animal-Free Block and Diluent is supplied without any R.T.U. Animal-Free Block and Diluent SP-5035
phosphatase activity. Our Normal Sera are pooled samples collected from healthy sodium azide. It can therefore be used with both peroxidase
adult animals. The serum is heat-treated and centrifuged to and alkaline phosphatase antibody conjugates and all Animal-Free Blocker™ SP-5030
• Can be added to the alkaline phosphatase substrate remove precipitates and then filtered. Each serum is tested
solution secondary detection reagents including polymer systems and
with the appropriate antibody to check for possible cross- avidin/biotin reagents that incorporate these enzymes. This
• Does not inhibit the isoenzyme used for the VECTASTAIN® reactivities. The sera can be used to block non-specific binding makes the blocking solution especially convenient in multiple
ABC-AP reagents, ImmPRESS™- AP Reagents and other or as an antibody diluent. antigen labeling IHC applications in which antibodies from
alkaline phosphatase conjugates different species and a variety of detection systems are used
• Ready-to-use in a convenient dropper bottle on the same tissue section.

36 vectorlabs.com vectorlabs.com 37
 Secondary and Tertiary Detection Reagents
Our secondary antibodies are prepared by hyper-immunizing animals in a manner that produces high affinity antibodies. These Biotinylated Unconjugated
are then purified by an affinity chromatography procedure designed to remove any low-affinity antibodies. Cross-reactivities Host Species (Concentrate) Host Species (Concentrate)
that can interfere with specific labeling are removed by solid-phase adsorption techniques. The final product is then subjected
Anti-Human Secondary Antibodies Goat Goat
to rigorous quality-control assays including immunodiffusion, solid-phase enzyme immunoassays, gel electrophoresis, solid-
Anti-Human IgG (H+L) BA-3000 AI-3000
phase binding assays and IHC tissue staining. These unconjugated antibodies are used to generate our enzyme conjugated and
Anti-Human IgE, ε (Epsilon) chain specific BA-3040 AI-3040
biotinylated secondary antibodies.
Anti-Human IgG, γ (Gamma) chain specific BA-3080 AI-3080

Biotinylated and Unconjugated Secondary Antibodies Anti-Human IgM, μ (Mu) chain specific BA-3020 AI-3020
Our high-affinity, purified, biotinylated and unconjugated secondary antibodies are manufactured under controlled conditions Anti-Human κ (Kappa) Chain, kappa chain specific BA-3060 AI-3060
to retain maximum specificity and affinity. Our secondary antibodies are subjected to rigorous quality control assays and can be
used for tissue and cell staining, ELISAs, and blotting.

Biotinylated Unconjugated
Host Species (Concentrate) Host Species (R.T.U.)† Host Species (Concentrate)

Secondary Antibodies Goat Rabbit Horse Goat Horse Goat Rabbit Horse
Enzyme Conjugated Secondary Antibodies Avidin and Streptavidin Enzyme Conjugates
Anti-Cat IgG (H+L) BA-9000
Our high-affinity, purified antibodies are cross-linked with Our enzyme-conjugated avidin and streptavidin are suitable
alkaline phosphatase (AP) or horseradish peroxidase (HRP) for use in solid-phase assays, tissue/cell staining systems, and
Anti-Chicken IgG (H+L) BA-9010 of the highest specificity. Our conjugation method ensures blotting. The conjugates are produced in optimized ratios with
Anti-Goat IgG (H+L) BA-5000
the maximum preservation of enzyme activity and antibody enzymes of the highest specific activity. Covalent linkages are
specificity. Recommended applications include tissue staining, specifically chosen to provide stable, highly active conjugates.
Anti-Goat IgG (H+L) BA-9500 BP-9500 AI-5000 ELISAs, and blotting.
Anti-Guinea Pig IgG (H+L) BA-7000

Anti-Hamster IgG (H+L) BA-9100 AI-9100 Catalog Catalog

Product Number Product Number
Anti-Horse IgG (H+L) BA-8000
Alkaline Phosphatase Alkaline Phosphatase
Anti-Mouse IgG (H+L) BA-2000 BP-2000 AI-9200 Anti-Mouse IgG (H+L) made in horse AP-2000 Alkaline Phosphatase Streptavidin SA-5100
Alkaline Phosphatase labeled
Anti-Mouse IgG (H+L), rat adsorbed BA-2001 Alkaline Phosphatase Avidin D A-2100
Anti-Rabbit IgG (H+L) made in goat AP-1000
Anti-Mouse IgG (H+L) BA-9200 BP-9200 AI-2000 Peroxidase
Alkaline Phosphatase labeled
Horseradish Peroxidase Streptavidin, concentrate SA-5004
Anti-Mouse IgM (H+L), Mu chain specific BA-2020 Peroxidase
Horseradish Peroxidase Streptavidin, R.T.U. SA-5704
Anti-Rabbit IgG (H+L) BA-1000 BP-9100 AI-1000 Anti-Mouse IgG (H+L) made in horse PI-2000
Peroxidase labeled Horseradish Peroxidase Avidin D, concentrate A-2004
Anti-Rabbit IgG (H+L) BA-1100 BP-1100
Anti-Rabbit IgG (H+L) made in goat PI-1000 Horseradish Peroxidase Avidin D, R.T.U. A-2704
Anti-Rat IgG (H+L) BA-4000 AI-4000 Peroxidase labeled
Anti-Human IgG (H+L) made in goat PI-3000
Anti-Rat IgG (H+L), mouse adsorbed BA-4001 AI-4001
Peroxidase labeled
Anti-Rat IgG (H+L) BA-9400 BP-9400 Anti-Goat IgG (H+L) made in horse PI-9500
Peroxidase labeled
Anti-Rat IgG (H+L), mouse adsorbed BA-9401

Anti-Sheep IgG (H+L) BA-6000

Anti-Swine IgG (H+L) BA-9020

Universal Anti-Mouse/Rabbit IgG (H+L) BA-1400 BP-1400

Universal Pan-Specific Anti-Mouse/Rabbit/Goat IgG (H+L) BA-1300

†
Ready-to-use, prediluted stabilized solutions

38 vectorlabs.com vectorlabs.com 39
 Mounting Media
VectaMount™ Permanent Mounting Medium Mounting Media / Substrate Compatibility
VectaMount™ Mounting Medium is an optically clear
formula for permanently preserving histochemical stains or VectaMount™ VectaMount™
precipitable enzyme substrates in tissue sections or Permanent AQ Aqueous
cell preparations. Mounting Mounting
Substrate Medium Medium
• Non-aqueous
HRP
• Toluene- and xylene-free (low toxicity)
Vector® DAB • •
• Resin-based media
Vector® DAB-Ni •
• Odorless ImmPACT™ DAB • •
• Viscosity formulated for easy application and uniform
ImmPACT™ DAB EqV • •
spreading
Vector® VIP • Colon: • CD3 (rm), ImmPRESS™ Reagent (HRP) Anti-Rabbit IgG, ImmPACT™ SG
• Compatible with most horseradish peroxidase and alkaline Melanoma: S100 (rp), VECTASTAIN® Elite® ABC Kit, Vector® VIP (purple), Vector®
ImmPACT™ VIP Methyl Green counterstain (green). Note color contrast with brown pigments in (blue-gray) • Cytokeratin AE1/AE3 (m), ImmPRESS™ Reagent (HRP) Anti-Mouse IgG,
phosphatase substrates •
ImmPACT™ AMEC Red (red).
tissue.
• Dries clear with an ideal refractive index suitable for Vector® NovaRED™ •
high-resolution oil-immersion microscopy ImmPACT™ NovaRED™ •
Vector® SG • •
VectaMount™ AQ Aqueous Mounting Medium ImmPACT™ SG • •
VectaMount™ AQ Aqueous Mounting Medium preserves Vector® AEC •
the color and clarity of enzyme substrates whose reaction
ImmPACT™ AEC •
products are soluble in alcohol or other organic solvents.
Stained and mounted sections can be stored in a slide box at ImmPACT™ AMEC Red •
room temperature for at least two years without fading. Vector® TMB •
• Hard-setting AP
• Simple to use, requires no mixing Vector® Red • •
ImmPACT™ Vector® Red • •
Vector® Blue • • Cytospin of EBV+ cell line: Epstein-Barr virus nuclear antigen 1 (EBNA-1; rat), Tumor: • CD34 (m), VECTASTAIN® Universal ABC-AP Kit, Vector® Blue AP Substrate
Product Catalog Number ImmPRESS™ Reagent (HRP) Anti-Rat IgG, ImmPACT™ NovaRED™ (red). (Image (blue) • Cytokeratin 8/18 (m), VECTASTAIN® Universal ABC-AP Kit, Vector® Red AP
Vector® Black • courtesy of Dr. GM Reynolds, Centre for Liver Research, University of Birmingham, U.K.) Substrate (red).
VectaMount™ Permanent Mounting Medium H-5000 Vector® BCIP/NBT • •
VectaMount™ AQ Aqueous Mounting Medium H-5501

Breast carcinoma: • CD31 (m), ImmPRESS™ Anti-Mouse IgG HRP Reagent, Melanoma: Vimentin (rm), ImmPRESS™-AP Anti-Rabbit IgG Reagent, Vector® Blue
ImmPACT™ DAB EqV HRP Substrate (brown) • MRC1 (r), ImmPRESS™- AP Anti- AP Substrate (blue). Note color contrast with brown pigments in tissue.
Rabbit IgG Reagent, ImmPACT™ Vector® Red AP Substrate (magenta). Image
courtesy of Richard Allen, part of the Academic Unit of Inflammation and
Tumour Targeting headed by Professor Claire Lewis.

40 vectorlabs.com vectorlabs.com 41
 Accessory Reagents
VECTABOND™ Reagent Tissue Section Adhesive
VECTABOND™ Reagent chemically modifies the surface of
glass to form a highly adherent charged surface. This charge
significantly increases the adherence of both frozen and
paraffin-embedded tissue sections and cell preparations to
glass microscope slides and coverslips. Tissue sections will
remain attached even when subjected to the most extreme
conditions, such as high-temperature antigen retrieval and in
situ hybridization. VECTABOND™ Reagent treated slides can be
stored indefinitely.
ImmPrint™ Histology Pen
The ImmPrint™ Histology Pen is a permanent marking pen
designed for writing on glass microscope slides, tissue
cassettes, and most hard surfaces. Unlike other pens
commonly used for histology, the ImmPrint™ Pen has a
smooth writing tip that resists drying out.
• High-density, fast-drying, black ink
• Resistant to most organic solvents encountered in
histological applications

ImmEdge™ Hydrophobic Barrier Pen
The ImmEdge™ Pen is a hydrophobic barrier (PAP) pen for
immunohistochemistry and in situ hybridization. It provides a
water-repellent barrier that keeps reagents localized on tissue
specimens and prevents mixing of reagents when multiple
sections are mounted on the same slide.
• Heat-stable
• Insoluble in alcohol and acetone
• Stable for use with buffers with and without detergent
(Tween 20, Triton X-100, etc.)
• Completely removed by all commonly used xylene and
xylene-substitute clearing agents
• Contains no ozone-depleting solvents
• Compatible with both enzyme- and fluorescence-based
detection systems
Control Antibodies
These antibodies are IgG preparations for use as controls for
primary antibodies made in rabbit, mouse, rat, or goat. Each
has been purified from pooled serum of healthy adult animals
and contain a spectrum of the IgG subclasses. When applied
appropriately, these controls will help determine whether the
primary antibody staining signal is specific for the antigen or
whether staining is the result of non-specific adsorption of
primary antibody to tissue sites.
Antigen Unmasking Solutions
Breast Carcinoma: Without (left panel) and with (right panel) Citrate-based Antigen Unmasking Solution, Estrogen receptor (m), ImmPRESS™ Anti-Rabbit IgG Kit, DAB (brown)
Our Antigen Unmasking Solutions are highly effective at
substrate. Hematoxylin QS (blue) counterstain.
revealing antigens in formalin-fixed, paraffin-embedded tissue
sections when used in combination with a high temperature
treatment procedure. We offer two formulations of Antigen
Unmasking Solution: Citrate-based solution (pH 6.0) and
Tris-based solution (pH 9.0), each supplied as 100X
concentrated stocks.

Product Catalog Number

VECTABOND™ Reagent (Tissue Section Adhesive) SP-1800
ImmEdge™ Hydrophobic Barrier Pen H-4000
ImmPrint™ Histology Pen H-6100
Control Antibodies
Rabbit IgG I-1000
Mouse IgG I-2000
Rat IgG I-4000
Goat IgG I-5000
Antigen Unmasking Solutions
Citrate-based (100X) (pH 6.0) H-3300
Tris-based (100X) (pH 9.0) H-3301

Lymph Node: Without (left panel) and with (right panel) TRIS-based Antigen Unmasking Solution, Cyclin D1 (rm), ImmPRESS™ Anti-Rabbit IgG Kit, DAB (brown) substrate.
Hematoxylin QS (blue) counterstain.

42 vectorlabs.com vectorlabs.com 43
 Contact Details
Ordering Information
Order online at: www.vectorlabs.com
Orders may also be placed by mail, telephone, fax, email or
secure online ordering. Please include the following with
each order:
• Product name and catalog number
• Unit size and quantity
• Billing and shipping addresses
• Purchase order number
• Name, phone number, address and email address of person
placing order

Orders using VISA, Mastercard, or American Express are

US Office accepted and processed immediately. Telephone orders
USA Headquarters: over $2000 may require written faxed confirmation.
Vector Laboratories, Inc. A confirmation should be boldly marked “Confirming Order.
30 Ingold Road Do Not Duplicate.” Duplicate shipments due to incorrectly
Burlingame, CA 94010 marked confirming orders cannot be returned for credit.
Tel: +1 (650) 697-3600 No returned product will be accepted or credited without
Ordering and Technical Service (USA): +1 (800) 227-6666 prior authorization from Vector Laboratories.
Fax: +1 (650) 697-0339 Please contact us to discuss discounts for custom or
Email: (USA) vector@vectorlabs.com large orders.
Email: (outside USA) intl@vectorlabs.com
Payment / shipping terms:
UK Office Payment terms: net 30 days. Prices are FCA Burlingame,
United Kingdom: California. Shipping charges will be prepaid and added to
Vector Laboratories Ltd., the invoice. Orders are usually shipped the same day they
3 Accent Park, Bakewell Road, Orton Southgate, are received. Unless requested otherwise, all products are
Peterborough, PE2 6XS shipped 2nd-day air. RCA60 products are shipped in the USA
United Kingdom only according to federal transportation regulations requiring
Tel: +44 (0) 1733 237999 additional shipping charges. We require a written confirmation
Email: vector@vectorlabs.co.uk for all RCA60 products.
Email: technical@vectorlabs.co.uk
International Orders
Vector products are also available from over 50 distributors
worldwide. Please contact us or visit our website for a
complete listing of these representatives.

Although provided in a highly purified form, our products are not intended for clinical diagnosis or drug use, nor have they been packaged under sterile conditions.
All products listed in this catalog are for research purposes only. The listing of any product in this catalog does not imply the absence of a patent covering its use, does
not constitute license under any existing or pending patent, nor is it intended or implied as a recommendation for the use of such products in infringement of any patent.
The responsibility for determining the existence of such patents rests solely with the user.

Trademarks of Vector Laboratories: ABC-AmP, Animal-Free Blocker, AT-Yellow, BLOXALL, DuoLuX, Elite, EndTag, FastTag, Fusion Aid, GC-Red, HYBEX, ImmEdge, ImmPACT, Human kidney tissue demonstrating a glomerulus (a cluster of blood vessels;
ImmPRESS, ImmPrint, M.O.M., Neurobiotin, NicKit p.s.o., Photoprobe, PolyBlock, ProFusion, ProtOn, Quant*Tag, Resolve-It, UltraSNAP, VECTABOND, VectaCell, VectaMount, brown), which is surrounded by various tubules as well as associated smooth
VECTASHIELD, VECTASHIELD HardSet, VECTASTAIN, Vector, Vector Black, Vector Blue, Vector NovaRED, Vector Red, Vectrex. muscle cells (red). Together these represent the ‘basic filtration unit’ of the
kidney. Image kindly supplied by Steffen Rickelt, David H. Koch Institute for
©Vector Laboratories, Inc. 2017 Integrative Cancer Research, Massachusetts Institute of Technology (MIT).

44 vectorlabs.com vectorlabs.com
 vectorlabs.com
vectorlabs.com